Oxidative stress contributes to a number of human degenerative diseases associated with ageing, such as Parkinson's disease, and Alzheimer's disease, as well as to Huntington's Chorea and Friedreich's Ataxia, and to non-specific damage that accumulates with aging. It also contributes to inflammation and ischaemic-reperfusion tissue injury in stroke and heart attack, and also during organ transplantation and surgery. To prevent the damage caused by oxidative stress a number of antioxidant therapies have been developed. However, most of these are not targeted within cells and are therefore less than optimally effective. Moreover, many such antioxidants have unfavourable physicochemical properties that limit for example, their bioavailability, and their ability to penetrate to the target organ to exert a therapeutic effect.
Mitochondria are intracellular organelles responsible for energy metabolism. Consequently, mitochondrial defects are damaging, particularly to neural and muscle tissues which have high energy demands. They are also the major source of the free radicals and reactive oxygen species that cause oxidative stress inside most cells. Therefore, the applicants believe delivering antioxidants selectively to mitochondria will be more effective than using non-targeted antioxidants. Accordingly, it is towards the provision of antioxidants which may be targeted to mitochondria that the present invention is directed.
Lipophilic cations may be accumulated in the mitochondrial matrix because of their positive charge (Rottenberg, 1979 Methods Enzymol 55, 547. Chen, 1988 Ann Rev Cell Biol 4, 155). Such ions are accumulated provided they are sufficiently lipophilic to screen the positive charge or delocalise it over a large surface area, also provided that there is no active efflux pathway and the cation is not metabolised or immediately toxic to a cell.
The focus of the invention is therefore on an approach by which it is possible to use the ability of mitochondria to concentrate specific lipophilic cations to take up linked antioxidants so as to target the antioxidant to the major source of free radicals and reactive oxygen species causing the oxidative stress.
Examples of antioxidant compounds that show good antioxidant activity yet exhibit poor bioavailability with respect to the target compartment in vivo include Coenzyme Q (CoQ) and Idebenone. Both of these compounds must be administered at very high dose rates to be efficacious, and therefore have low therapeutic efficacy when referenced to the dose rate administered.
We believe without wishing to be bound by any theory that for an antioxidant compound, activity in vitro or ex vivo (such as, for example, antioxidant activity or mitochondrial accumulation) is by no means the sole determinant of efficacy in vivo (such as, for example, therapeutic efficacy). Whilst it is true that to be useful as a mitochondrially targeted antioxidant compound of the present invention, an antioxidant compound must exhibit a suitable antioxidant activity in vitro or ex vivo, to be efficacious in vivo the mitochondrially targeted antioxidant compound must exhibit other desirable physicochemical properties, such as, for example, suitable bioavailability, localization or distribution within the target mitochondria, and/or suitable stability.
We believe without wishing to be bound by any theory that, at least in part by virtue of their physicochemical properties, such as, for example, their amphiphilicity and/or low partition coefficient, the mitochondrially targeted antioxidant compounds of the present invention exhibit advantageous antioxidant functionality, including bioavailability, and/or mitochondrial targeting and accumulation in vivo. Such compounds are thereby therapeutically efficacious at low dose rates in comparison to other antioxidant compounds.
In U.S. Pat. No. 6,331,532 by reference to exemplifications of compounds mitoquinol and mitoquinone (referred to collectively herein as mitoquinone/mitoquinol) there is disclosed the prospect of mitochondrial targeting of an antioxidant moiety reliant upon a lipophilic cation covalently coupled to the antioxidant moiety. The exemplified compound therein (despite generalisation of the bridge length), is the compound mitoquinone of the formula
with a carbon bridge length of 10 (i.e. C10 bridged). Its reduced form, mitoquinol, is also C10 bridged.
Mitoquinone/mitoquinol, despite excellence in antioxidant activity and targeting and accumulation in mitochondria in vitro and in vivo, we have found to be somewhat unstable as the bromide salt.
We have also determined that mitoquinone (1)/mitoquinol has a moderately high partition coefficient (e.g. about 160 when assessed by an octanol:water partition system, see herein), that idebenone has a high partition coefficient of 3.1×103, and ubiquinone (CoQ10) has a very high partition coefficient of 1.8×1020.
We believe compounds of the general formula I
where the bridge length is less than about C20 (for example less than about C15, in other examples less than about C10, and in other examples less than about C7) surprisingly may provide in vivo antioxidant activity at or in the mitochondria many times beyond what would be expected from the in vitro or ex vivo studies of antioxidant activity, including mitochondrial targeting of antioxidant activity, and many times beyond that observed with, for example, derivatives of CoQ such as CoQ0.
A lower partition coefficient is in our view desirable for particular applications, and may provide for greater bioavailability, particularly where administration is to be oral or parenteral and/or where there is targeting of the antioxidant compound to mitochondria in the tissues of internal organs (e.g. brain, heart or other organs). Conversely, we believe compounds which exhibit high partition coefficients may be less appropriate for delivery orally for the treatment of oxidative stress where there is a requirement for oral bioavailability, organ penetration, and for passage through a barrier such as that of the blood brain barrier.
In PCT/NZ02/00154 there is disclosed a process for manufacturing compounds of the general formula II

Such a procedure we have found is more suitable for chain lengths or for bridging groups greater than C6 (i.e. where n>6) than it is for chain lengths or for bridging groups less than C6 (i.e. where n<6).
The present invention recognises therefore an advantage in being able to prepare compounds of bridge length C6 or less.
We have found on preparation of compounds of bridge length less than about C10 that such compounds have distinct and desirable physicochemical properties from those taught within U.S. Pat. No. 633,152 and PCT/NZ02/00154.
Advantageously, examples of compounds of the present invention are crystalline and/or solid in form, which amongst other advantages renders them particularly suitable to formulation (e.g. by tableting or encapsuling) in pharmaceutical formulations for example in orally-administerable dosage forms. This results in novel mitochondrially targeted antioxidant compounds with desirable physicochemical properties particularly suited to therapeutic pharmaceutical use.
Other examples of compounds of the present invention may be in a form other than a crystalline and/or solid form, but may be amenable to formation of a solid form by admixture with other agents such as for example, carriers, excipients, complexation agents, or other additives and the like, such as, for example, cyclodextrins. Advantageously such agents are pharmaceutically acceptable.
We have also determined a desirability to offer examples of the amphiphilic mitochondrially targeted antioxidant compounds of the present invention with their positive charge in association with a suitable anion thereby to provide the compound as a general neutralised salt form, including but not limited to solid or crystalline products. In such salt forms however certain salt forming anions we have found to be best avoided as they exhibit reactivity against the antioxidant compound, for example, against the antioxidant moiety, the linking moiety, or the lipophilic cationic moiety, and/or may lead to cleavage at or of the antioxidant moiety. Other salt forming anions are considered pharmaceutically undesirable. For example, nitrate moieties are considered inappropriate generally by pharmaceutical companies as being pharmaceutically or environmentally unacceptable, whilst a hydrogen bromide frequently used in salt forming of such compounds we find to have nucleophilic properties that can lead to a reactivity against the antioxidant moiety, for example, a cleavage of a methyl group from the antioxidant moiety of the compound of general formula (II) herein, and/or some overall decrease in stability of the overall compound. For example, we have determined that the bromide salt of compound Mitoquinone is somewhat unstable.
We believe therefore that salt forms, including salt forms as a solid or crystalline form, of mitochondrially targeted antioxidants are best associated with an anion or like moiety that is not nucleophilic, or one which does not exhibit reactivity against any of the moieties comprising the antioxidant compound or complex. It is also preferable that the anion is pharmaceutically acceptable.
We further believe, without wishing to be bound by any theory, that at least in part by virtue of their physicochemical properties, the antioxidant compounds of the present invention are able to selectively target antioxidant activity to particular intracellular and/or intramitochondrial locations, for example so as to target particular reactive oxygen species, and/or particular sites of reactive oxygen species generation.